CN114574512A - Preparation of cell-penetrating peptide-target protein compound and method for efficiently introducing cell-penetrating peptide-target protein compound into streptomyces living cells - Google Patents
Preparation of cell-penetrating peptide-target protein compound and method for efficiently introducing cell-penetrating peptide-target protein compound into streptomyces living cells Download PDFInfo
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Abstract
The invention discloses a preparation method of a cell-penetrating peptide-target protein compound and a method for efficiently introducing the cell-penetrating peptide-target protein compound into a streptomyces living cell. The method can help target exogenous protein to efficiently cross complex cell walls and cell membrane barriers of gram-positive bacteria, and is helpful for mediating exogenous protein to efficiently enter streptomycete living cells through the method so as to carry out living cell level or visual level research on cell gene expression regulation, metabolite yield monitoring and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a preparation method of a cell-penetrating peptide-target protein compound and a method for efficiently introducing the cell-penetrating peptide-target protein compound into streptomyces living cells.
Background
Cell Penetrating Peptides (CPPs) are short peptides consisting of about 5 to 30 amino acids, which can serve as living Cell mass transport systems, carry proteins, nucleic acids, nanomaterials and the like of interest through physiological barrier Cell membranes of animal cells in a covalent or non-covalent coupling mode, penetrate the Cell membranes of the animal cells, and have small influence on Cell activities within a certain concentration. Currently, CPPs have been widely used in noninvasive drug delivery tools targeting intracellular molecules. Recent researches show that the CPP not only can overcome cell membranes of animal cells by carrying oligonucleotides, small interfering RNAs, plasmids, nano materials and the like, but also can carry the small interfering RNAs, the plasmids and the like to higher plants, algae and microbial cells containing cell wall structures, and related reports are less frequent.
Currently, the existing research on the transfer of 'goods' mediated by cell-penetrating peptides to living cells of microorganisms focuses on the cell-penetrating peptides, so that the uptake of antibacterial active substances such as antibacterial peptides, peptide nucleic acids and functionalized photodynamic antibacterial nanoparticles by the cells of the microorganisms can be increased, and the function of enhancing the antibacterial effect is achieved.
In addition, in recent years, researchers have begun to apply cell-penetrating peptides to the microbial cell carriage of plasmids and nanomaterials. In 2018, transfer of the derivative cell-penetrating peptide and nanogold conjugate based on Tat to escherichia coli and staphylococcus aureus is realized for the first time by Munish Kumar. In 2019, Md Monirul Islam et al firstly passed the 205 kb plasmid pMSR227 containing fluorescent protein mCherry reporter gene and cell-penetrating peptide (KH)9The means of incubating the cells directly after non-covalent coupling of BP100 will successfully beThe plasmid was transformed into E.coli DH 5. alpha. cells, and it was confirmed that the transformation efficiency of the method was similar to that of electroporation and was significantly higher than that of the heat shock method.
As can be seen from the above description, cell-penetrating peptides have begun to be applied to live cell delivery of foreign substances to microbial cells, which has the potential for use in carrying a variety of foreign substances into live cells of microbes. However, at present, no relevant research and application report of introducing exogenous protein into streptomyces living cells mediated by cell-penetrating peptide is found.
Disclosure of Invention
The invention aims to provide a method for efficiently introducing exogenous protein into streptomyces living cells and a preparation method of a cell-penetrating peptide-target protein compound related to the method. The introduction of the target protein into the streptomycete living cells by utilizing the cell-penetrating peptide can be applied to aspects of streptomycete metabolite detection, gene expression regulation, cell function regulation, biological control and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing a cell-penetrating peptide-target protein complex, comprising the steps of: the method comprises the following steps:
(1) constructing a CPP-mCherry protein expression plasmid pET28 a-CPP-mChery: constructing a fusion expression gene of the cell-penetrating peptide, 4 glycine connecting peptides and the fluorescent protein mCherry in an escherichia coli expression plasmid pET-28a (+);
(2) the plasmid pET28a-CPP-mCherry is transformed into Escherichia coli by calcium chloride chemical conversion methodE.coliBL21(DE3) competent cells;
(3) inoculating CPP-mCherry recombinant expression strain into 5mL LB liquid culture medium containing kanamycin, activating at 37 ℃ and 220rpm for 8h, inoculating the activated strain into 250mL 2 XYT culture medium at a volume ratio of 1:100, adding 250 μ L kanamycin (concentration 50-55 mg/mL), and culturing for 4.5-5.5h until OD is reached600When the value reaches 0.6-0.8, cooling the bacterial liquid to 16 ℃, adding IPTG (isopropyl-beta-thiogalactoside) and carrying out induced expression at 16 ℃ and 220rpm for 16-20 h;
(4) purifying and concentrating the CPP-mCherry fusion protein: centrifuging the bacterial liquid for induced expression, collecting thalli, adding 30mL of PBS solution into the thalli, stripping the thalli from the wall of a centrifugal bottle by using a glass rod, then thoroughly resuspending the thalli on a vortex instrument (the thalli obtained by 500mL of the bacterial liquid is dissolved in 30mL of PBS at most), ultrasonically crushing the resuspended thalli, then transferring the crushed bacterial liquid into a 50mL centrifugal tube, balancing, centrifuging at 4 ℃ and 10000 rpm for 30 min, and collecting supernatant; after the cobalt affinity chromatography column is balanced by 3-5 column volumes through PBS, the collected supernatant is passed through the column, and the column hanging is repeated once; washing 3-5 column volumes with PBS for removing impurities such as cell debris which cannot be removed by high-speed centrifugation; washing 3-5 column volumes with PBS containing 5mM imidazole for removing the non-specifically bound hetero-proteins from the gel; washing 5-8 column volumes with PBS containing 150mM imidazole to dissociate the target protein bound specifically to the gel, and collecting all eluates; washing 3-5 column volumes with PBS containing 500mM imidazole, dialyzing the collected protein, and concentrating to obtain the cell-penetrating peptide-target protein recombinant fusion protein.
(5) Cobalt affinity chromatography column renaturation: first use ddH2O-wash 6-8 column volumes, then wash 2 column volumes with 0.4N NaOH, immediately with ddH2O-Wash 6-8 column volumes, followed by 0.1M EDTA and ddH2After washing 6-8 column volumes with O, 0.1M CoCl was immediately applied2Wash 5-8 column volumes and incubate for 15min at room temperature; incubation completed with large amount of ddH2After O washing, washing 3-5 columns with PBS, balancing the volume, and storing for a long time at 4 ℃.
Further, the cell-penetrating peptide and the amino acid sequence thereof in the step (1) are one of the following:
Tat：YGRKKRRQRRR；
ANTP：RQIKIWFQNRRMKWKK；
KFF3K：KFFKFFKFFK；
R9：RRRRRRRRR；
K9：KKKKKKKKK；
KH9-BP100：KHKHKHKHKHKHKHKHKHKKLFKKILKYL；
the cell-penetrating peptide-target protein compound correspondingly obtained in the step (4) is Tat-mCherry, ANTP-mCherry (KFF)3K-mCherry、R9-mCherry、K9-mCherry、(KH)9-BP 100-mCherry.
In the step (3), the concentration of kanamycin in the LB liquid culture medium is 50-55 mu g/mL, and the final concentration of IPTG is 0.1-0.4 mM.
In the step (4), the ultrasonic crushing time of the resuspended thalli is 15min, the ultrasonic treatment is carried out for 2s, the pause is 4s, and the ultrasonic treatment process is operated in a dark environment.
In the step (4), the specific steps of protein dialysis and concentration are as follows: putting the collected protein into a dialysis bag, immersing the dialysis bag in a PBS solution to replace imidazole in the protein collection solution, and keeping the dialysis device at 4 ℃ overnight in a dark place because the target protein has fluorescence; changing the dialysate the next day; placing the dialyzed protein in a protein concentration tube, centrifuging at 4 ℃ and 4000 g, and concentrating the protein to 1.5-2 mL with the concentration of about 150 mu M; the whole experimental operation is protected from light; the resulting protein was stored frozen at-80 ℃ in the dark.
A high-efficiency streptomycete living cell exogenous protein introduction method comprises the following steps:
(1) and (3) treating streptomycete spores: adding 4 mL of PBS solution on a streptomycete plate, then using an inoculating loop to lightly scrape streptomycete spores, using a pipette to blow and uniformly mix, uniformly distributing the bacterial liquid into a centrifugal tube, centrifuging for 3 min at 4000 rpm, collecting thalli, washing for 2 times by using PBS, and then absorbing redundant solution to obtain streptomycete sporophytes;
(2) respectively adding Tat-mCherry, ANTP-mCherry (KFF) into the same streptomycete sporophyte3K-mCherry、K9-mCherry、R9-mCherry、(KH)9Uniformly diluting BP100-mCherry to 50 μ M with PBS, taking 100 μ L of diluent, adding to cells, gently mixing, incubating at 34 ℃ in the dark for 1h, and taking 50 μ M of fluorescent protein mCherry without the cell-penetrating peptide segment as a control;
(3) after incubation, centrifuging at 10000 rpm for 1 min to remove supernatant, adding 500 mu L PBS for resuspension and washing once, centrifuging to remove supernatant, adding 100 mu L0.25% trypsin, and digesting at 37 ℃ for 3-5 min to remove protein adsorbed on the surface of the thallus; after removing trypsin by centrifugation and washing twice with PBS, the mycelia were resuspended in 500. mu.L of PBS.
The method provided by the invention realizes the efficient loading of the exogenous protein into the streptomycete living cells, and is beneficial to the research of physiological condition research on streptomycete metabolic pathways by taking the protein as an element, the intervention regulation and control on streptomycete gene expression at the living cell level, the detection of metabolites at the living cell level, the fluorescence labeling visualization and the like.
The invention can realize the purpose of efficiently transferring the exogenous target protein to the living cells of the streptomycete, and has no obvious influence on the activity of the cells. The method can help target exogenous protein to efficiently cross complex cell walls and cell membrane barriers of gram-positive bacteria, and is helpful for mediating exogenous protein to efficiently enter streptomycete living cells through the method so as to carry out living cell level or visual level research on cell gene expression regulation, metabolite yield monitoring and the like.
Drawings
FIG. 1 is a schematic diagram of the introduction of exogenous target protein into Streptomyces live cells mediated by cell-penetrating peptide of the present invention.
FIG. 2 is a graph showing fluorescence intensity of 50. mu.M of different cell-penetrating peptide-mCherry fusion proteins in live Streptomyces cells in step (4) of example 2 of the present invention.
FIG. 3 is the statistics of fluorescence intensity of 50 μ M different cell-penetrating peptide-mCherry fusion proteins in live Streptomyces cells in step (5) of example 2 of the present invention.
FIG. 4 is the statistics of the cell-entering efficiency of 50 μ M different cell-penetrating peptide-mCherry fusion proteins in the live Streptomyces cells in step (5) of example 2 of the present invention.
FIG. 5 shows the different concentrations of cell-penetrating peptide (KH) in step (5) of example 2 of the present invention9-statistics of fluorescence intensity of BP100-mCherry fusion protein in live Streptomyces cells.
FIG. 6 shows the different concentrations of cell-penetrating peptide (KH) in step (5) of example 2 of the present invention9Statistics of the cell entry efficiency of the BP100-mCherry fusion protein in live streptomycete cells.
FIG. 7 shows the different concentrations of cell-penetrating peptide (KH) in step (5) of example 2 of the present invention9-effect of BP100-mCherry fusion protein on streptomyces cell activity.
Detailed Description
The following describes a method for efficiently introducing exogenous protein into streptomycete living cells, verification of carrying effect thereof, and a method for preparing fusion protein related to the method in detail by specific examples. Unless otherwise indicated, the reagents and assays referred to below are all conventional in the art.
Example 1
The preparation method of the cell-penetrating peptide-target protein complex comprises the following steps:
(1) constructing a CPP-mCherry protein expression plasmid pET28 a-CPP-mChery: the cell-penetrating peptide, 4 glycine connecting peptides and a fluorescent protein mCherry fusion expression gene are constructed in an escherichia coli expression plasmid pET-28a (+), and the amino acid sequences of the cell-penetrating peptide and the cell-penetrating peptide are tat (YGRKKRRQRRR), ANTP (RQIKIWFQNRRMKWKK) and KFF3K（KFFKFFKFFK）、R9（RRRRRRRRR）、K9（KKKKKKKKK）、KH9-BP100(KHKHKHKHKHKHKHKHKHKKLFKKILKYL)。
(2) The plasmid pET28a-CPP-mCherry is transformed into Escherichia coli by calcium chloride chemical conversion methodE.coliBL21(DE3) competent cells.
(3) Inoculating the CPP-mCherry recombinant expression strain into 5mL LB liquid culture medium containing 50 ug/mL Kanamycin (Kanamycin, Kan), activating at 37 ℃ and 220rpm for 8h, inoculating the activated strain into 250mL 2 XYT culture medium at a volume ratio of 1:100, adding 250 ul Kan, culturing for about 5h, and waiting until OD is reached600The value reaches about 0.6-0.8, the bacterial liquid is cooled to 16 ℃, IPTG (final concentration is 0.1 mM) is added, and the induction expression is carried out for 16-20 h at 16 ℃ and 220 rpm.
(4) Purifying and concentrating the CPP-mCherry fusion protein: centrifuging the induced expression bacterial liquid at 6000 rpm at 4 ℃ for 15min to collect thalli, adding 30mL of PBS solution into the thalli, stripping the thalli from the wall of a centrifugal bottle by using a glass rod, and then completely suspending the thalli on a vortex instrument (the thalli obtained by 500mL of bacterial liquid is dissolved in 30mL of PBS at most); carrying out ultrasonic crushing on the resuspended thalli for 15min, carrying out ultrasonic treatment for 2s, and carrying out stop for 4s, wherein the operation is carried out in a dark environment in the ultrasonic treatment process. Transferring the crushed bacterial liquid into a 50mL centrifuge tube, balancing, centrifuging at 4 ℃ and 10000 rpm for 30 min, and collecting supernatant; after the Co column is balanced by 3-5 column volumes through PBS, the collected supernatant is passed through the column and the column hanging is repeated once; washing 3-5 column volumes with PBS for removing impurities such as cell debris which cannot be removed by high-speed centrifugation; washing 3-5 column volumes with PBS containing 5mM imidazole for removing the non-specifically bound hetero-proteins from the gel; washing 5-8 column volumes with PBS containing 150mM imidazole to dissociate the target protein bound specifically to the gel, and collecting all eluates; washing 3-5 column volumes with PBS containing 500mM imidazole, loading the collected protein into a dialysis bag, immersing in PBS solution to displace imidazole in the collection; because the target protein has fluorescence, the dialysis device is kept at 4 ℃ overnight in a dark place; changing the dialysate the next day; placing the dialyzed protein in a protein concentration tube, centrifuging at 4 ℃ and 4000 g, and concentrating the protein to 1.5-2 mL with the concentration of about 150 mu M; the whole experimental operation is protected from light; the resulting protein was stored frozen at-80 ℃ in the dark.
(5) Cobalt affinity chromatography column renaturation: first use ddH2Washing 6-8 column volumes with O, and then washing 2 column volumes with 0.4N NaOH; ready-to-use ddH2O-Wash 6-8 column volumes, followed by 0.1M EDTA and ddH2After washing 6-8 column volumes with O, 0.1M CoCl was immediately applied2Wash 5-8 column volumes and incubate for 15min at room temperature; incubation completed with large amount of ddH2After washing, washing 3-5 columns with PBS, balancing the volume, and storing for a long time at 4 ℃.
Example 2
A foreign protein introduction method suitable for streptomycete living cells comprises the following steps:
(1) and (3) treating streptomycete spores: adding 4 mL of PBS solution on a streptomycete plate, then using an inoculating loop to lightly scrape off streptomycete spores, using a pipette to blow and mix the streptomycete spores uniformly, evenly dividing the bacterial liquid into a centrifugal tube, centrifuging the bacterial liquid for 3 min at 4000 rpm, collecting thalli, washing the thalli for 2 times by using PBS, and then absorbing the redundant solution to obtain streptomycete sporophytes.
(2) Taking the same amount of bacteria, adding Tat-mCherry, ANTP-mCherry (KFF)3K-mCherry、K9-mCherry、R9-mCherry、(KH)9-BP100-mCherryThen, the mixture was diluted to 50. mu.M with PBS. Adding 100 μ L of the above dilution to the cells, mixing gently, incubating at 34 deg.C in the dark for 1 h; the fluorescent protein mCherry with 50. mu.M containing no membrane-penetrating peptide fragment was used as a control.
(3) After incubation, centrifuging at 10000 rpm for 1 min to remove supernatant, adding 500 mu L PBS for resuspension and washing once, centrifuging to remove supernatant, adding 100 mu L0.25% trypsin, and digesting at 37 ℃ for 3-5 min to remove protein adsorbed on the surface of the thallus; after removing trypsin by centrifugation and washing twice with PBS, the mycelia were resuspended in 500. mu.L of PBS.
(4) Fluorescence microscopy (all samples were inserted on ice protected from light before preparation of the observation samples): the microscope was started 20 min in advance and warmed up, and 5. mu.L of the resuspended suspension was pipetted onto a glass slide with a pipette, covered with a glass slide, and examined under a fluorescence microscope (FIG. 2).
(5) The efficiency and the fluorescence intensity of different cell-penetrating peptide mediated fluorescent protein mCherry cells are counted, the optimal cell-penetrating peptide applicable to streptomycete in the tested range is screened, the cell-penetrating condition of the cell-penetrating peptide mediated fluorescent protein is optimized, the influence of the cell-penetrating peptide on the activity of streptomycete cells is investigated, and the result is shown in figures 3-7.
Claims (7)
1. A method for preparing a cell-penetrating peptide-target protein complex, which comprises the following steps: the method comprises the following steps:
(1) constructing a CPP-mCherry protein expression plasmid pET28 a-CPP-mChery: constructing a fusion expression gene of the cell-penetrating peptide, 4 glycine connecting peptides and the fluorescent protein mCherry in an escherichia coli expression plasmid pET-28a (+);
(2) transforming the plasmid pET28a-CPP-mCherry into E.coli BL21(DE3) competent cells by using a calcium chloride chemical transformation method;
(3) inoculating CPP-mCherry recombinant expression strain into 5mL LB liquid medium containing kanamycin, activating at 37 deg.C and 220rpm for 8h, inoculating the activated strain into 250mL 2 XYT medium at a volume ratio of 1:100, adding 250 μ L kanamycin with a concentration of 50-55mg/mL, and culturing for 4.5-5.5h until OD is reached600The value reaches 0.6-0.8, and the bacterial liquid is cooled to 16 DEG CAdding IPTG, and carrying out induced expression for 16-20 h at 16 ℃ and 220 rpm;
(4) purifying and concentrating the CPP-mCherry fusion protein: centrifuging the bacteria liquid for induced expression, collecting thalli, adding 30mL of PBS (phosphate buffer solution) into the thalli, peeling off the thalli, then thoroughly suspending the thalli on a vortex instrument, ultrasonically crushing the suspended thalli, centrifuging the crushed bacteria liquid, and collecting supernatant; after the cobalt affinity chromatographic column is balanced by 3-5 column volumes through PBS, the collected supernatant passes through the column, and the column hanging is repeated once; washing 3-5 column volumes with PBS, washing 3-5 column volumes with PBS containing 5mM imidazole, washing 5-8 column volumes with PBS containing 150mM imidazole, and collecting all eluates; washing 3-5 column volumes with PBS containing 500mM imidazole, dialyzing the collected protein, and concentrating to obtain the cell-penetrating peptide-target protein recombinant fusion protein.
2. The method according to claim 1, wherein the step of preparing the cell-penetrating peptide-target protein complex comprises: the cell-penetrating peptide and the amino acid sequence thereof in the step (1) are one of the following:
Tat：YGRKKRRQRRR；
ANTP：RQIKIWFQNRRMKWKK；
KFF3K：KFFKFFKFFK；
R9：RRRRRRRRR；
K9：KKKKKKKKK；
KH9-BP100：KHKHKHKHKHKHKHKHKHKKLFKKILKYL；
the cell-penetrating peptide-target protein compound correspondingly obtained in the step (4) is Tat-mCherry, ANTP-mCherry (KFF)3K-mCherry、R9-mCherry、K9-mCherry、(KH)9-BP 100-mCherry.
3. The method according to claim 1, wherein the step of preparing the cell-penetrating peptide-target protein complex comprises: in the step (3), the concentration of kanamycin in the LB liquid culture medium is 50-55 mu g/mL, and the final concentration of IPTG is 0.1-0.4 mM.
4. The method according to claim 1, wherein the step of preparing the cell-penetrating peptide-target protein complex comprises: in the step (4), the ultrasonic crushing time of the resuspended thalli is 15min, the ultrasonic treatment is carried out for 2s, the pause is 4s, and the ultrasonic treatment process is operated in a dark environment.
5. The method according to claim 1, wherein the step of preparing the cell-penetrating peptide-target protein complex comprises: in the step (4), the specific steps of protein dialysis and concentration are as follows: and (3) putting the collected protein into a dialysis bag, immersing the dialysis bag in a PBS solution, dialyzing overnight at 4 ℃ in a dark place, putting the dialyzed protein into a protein concentration tube, centrifuging, concentrating the protein to the concentration of 150 mu M, and keeping the whole experimental operation in the dark place.
6. The method according to claim 1, wherein the step of preparing the cell-penetrating peptide-target protein complex comprises: further comprising the step (5) of carrying out cobalt affinity chromatography column renaturation: by ddH2O-wash 6-8 column volumes, then wash 2 column volumes with 0.4N NaOH, and immediately ddH2O-wash 6-8 column volumes, followed by 0.1M EDTA, ddH2After washing 6-8 column volumes with O, 0.1M CoCl was immediately applied2Wash 5-8 column volumes and incubate at room temperature for 15min, complete incubation with large amount of ddH2After O wash, wash 3-5 column volumes with PBS to equilibrate.
7. A high-efficiency streptomycete living cell exogenous protein introduction method is characterized by comprising the following steps:
(1) and (3) treating streptomycete spores: adding a PBS solution on a streptomycete plate, scraping streptomycete spores by using an inoculating loop, blowing and uniformly mixing by using a pipette tip, uniformly distributing a bacterial liquid into a centrifugal tube, centrifugally collecting thalli, washing for 2 times by using PBS, and absorbing redundant solution to obtain streptomycete sporophytes;
(2) adding a cell-penetrating peptide-target protein compound into streptomycete sporophyte, wherein the cell-penetrating peptide-target protein compound is Tat-mCherry, ANTP-mCherry (KFF)3K-mCherry、K9-mCherry、R9-mCherry、(KH)9-BP100-mCherryDiluting to 50 mu M with PBS, taking 100 mu L of the diluent, adding the diluent to cells, uniformly mixing, and incubating for 1h at 34 ℃ in a dark place;
(3) after incubation, the supernatant was centrifuged and discarded, PBS was added for resuspension and washing once, the supernatant was centrifuged and discarded, 0.25% trypsin was added, digestion was carried out at 37 ℃ for 3-5 min, centrifugation was carried out, PBS was used for washing twice, and then the mycelia were resuspended in PBS.
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